The present invention relates to methods, devices and kits for use in screening patients to detect cancers and precancers associated with mucosal tissues, particularly oral cancer, using optical molecular imaging.
Cancer is defined as the uncontrollable growth of cells that invade and cause damage to surrounding tissue. Many properties of mammalian cells are either expressed at or mediated through the cell surface, and cancer is associated with characteristic molecular changes on the cellular surface.
Specifically, membrane proteins and lipids having attached carbohydrate side chains project from the external surface of a cell and form the cell glycocalyx. The glycosyl structures of these carbohydrate side chains vary among different cell types, among the same cells in different stages of the maturation process, and during pathological changes such as cancer (see, e.g., Ramsey I S, Delling M and Clapham D E, Annu Rev Physiol 2006; 68: 619-47; Gunthorpe M J and Chizh B A, Drug Discov Today 2009; 14: 56-67; Pankratov Y V, Lalo U V and Krishtal O A, J Neurosci 2002; 22: 8363-9). The side chains are heterogeneous in their oligosaccharide composition, length and branching status, conferring distinct biochemical and antigenic properties of the glycoconjugates, as, for example, with the idiotypic blood group antigens present on epithelial cells. These side chains also play an important role in regulating cell proliferation (see Feske S et al., Nature 2006; 441: 179-85; Cahalan M D, Nat Cell Biol 2009; 11: 669-77). Altered glycosylation of cell surface proteins, especially the terminal glycoprotein epitopes, may play a significant role in cell-cell interactions, development of cell adhesion, malignant transformation, and metastasis.
Altered glycosylation is a universal feature of cancer cells, and certain types of glycan structures are well-known markers for tumor progression (Varki A, Cummings, E. et al Editors, Essentials of Glycobiology, Cold Spring Harbor Laboratory Press Cold Spring Harbor, New York). Aberrant glycosylation occurs in essentially all types of experimental and human cancers, as has been observed for over 35 years, and many glycosyl epitopes constitute tumor-associated antigens. A long-standing debate is whether aberrant glycosylation is a result or a cause of cancer. Many recent studies indicate that some, if not all, aberrant glycosylation is a result of initial oncogenic transformation, as well as a key event in induction of invasion and metastasis.
Glycosylation promoting or inhibiting tumor cell invasion and metastasis is of crucial importance in current cancer research. Nevertheless, this area of study has received little attention from most cell biologists involved in cancer research, mainly because structural and functional concepts of glycosylation in cancer are more difficult to understand than the functional role of certain proteins and their genes in defining cancer cell phenotypes. Glycosylation appears to be considered “in the shade” of more popular topics such as oncogenes and antioncogenes, apoptosis, angiogenesis, growth factor receptors, integrins and adherins function, etc., despite the fact that aberrant glycosylation profoundly affects all of these processes (Senitiroh Hakomori, Glycosylation defining cancer malignancy: New wine in an old bottle. PNAS, Aug. 6, 2002, vol. 99, no. 16 10231-10233).
The carbohydrate side chains discussed above form receptor sites for lectins, which are non-enzymatic proteins or glycoproteins of non-immune origin that bind specifically and non-covalently to specific oligosaccharide chains (see Brandman O et al., Cell 2007; 131: 1327-39). Lectins can thus serve to identify cell types or cellular components and have become useful analytical tools for studying the altering of cell surface carbohydrates in disease stages (Roderick H L and Cook S J, Nat Rev Cancer 2008; 8: 361-75). During post-translational events and in the course of disease, including cancer, cellular proteins are modified, often by changing the glycosylation of the cellular proteins. Thus, lectins can be used as targets for identifying cell surface changes associated with cancers and other conditions.
Some lectins recognize oligosaccharides only, while others bind both oligosaccharides and monosaccharides (mannose, galactose/N-acetyl galactosamine, N-acetyl glucosamine, fucose, and sialic acid). Monosaccharide-specific lectins are usually classified according to one of five groups depending upon its highest-affinity monosaccharide.
Even so, most lectins interact with more than one group, albeit at a lower association constant, such that it can be difficult to associate binding of a particular lectin with a specific disease. Some lectins can interact with monosaccharides from different specificity groups through the same binding site. Other lectins can bind simultaneously with distinct sugars. Still others can combine with monosaccharides that appear structurally unrelated, but that present similar topographical features when appropriately viewed.
For example, WGA binds N-acetyl glucosamine and, more weakly, N-acetyl galactosamine. WGA also binds N-acetyl neuraminic acid in free form because this monosaccharide is structural similar to N-acetyl glucosamine. Many monosaccharide-specific lectins can be classified according to the type of protein-linked carbohydrate unit they recognize. As such, some lectins react primarily with O-linked sugar units, whereas others bind N-linked units.
Although each unique lectin has a higher specificity for certain groups, very few lectins bind only a single sugar unit and each lectin provides distinctly different background binding properties and signal-to-noise ratios in tissue.
Lectins are found in plants, animals, and fungi, and a variety of lectins are known in the art. The carbohydrate binding specificity of a number of commercially available lectins is well known in the art (see e.g. Lectin and Lectin Conjugates Catalog Addendum, EY Laboratories, Inc., San Mateo, Calif., 2010). A non-limiting list of commercially available plant lectins is shown by organism of origin and standard abbreviation in Table 1 below. In addition, new lectins are discovered each year.
TABLE 1Selected Commercially Available Lectins (adapted fromLectin and Lectin Conjugates Catalog Addendum, EYLaboratories, Inc., San Mateo, California, 2010).Lectin source (Latin name)Common nameAbbreviationAbrus precatoriusjequirity beanAPAAegopodium podagrariaground elderAPPAgaricus bisporusmushroomABAAllomyrina dichotomaJapanese beetleAllo AAnguilla Anguillafresh water eelAAAArachis hypogaeapeanutPNAArtocarpus integrifoliajackfruitJacalin AIABauhinia purpureacamel's foot treeBPABryonia dioicawhite bryonyBDACanavalia ensiformisjack beanCon ACancer antennariusCalifornia crabCCACaragana arborescenspea treeCAACicer arietinumchick pea, ceci beanCPAColchicum autumnalemeadow saffronCACytisus scopariusscotch broomCSADatura stramoniumjimson weedDSADolichos biflorushorse gramDBAErythrina cristagallicoral treeECAEuonymus europaeusspindle treeEEAGalanthus nivalissnowdropGNAGlycine maxsoybeanSBAGriffonia simplicifoliaGS-I, GS-IIHelix aspersagarden snailHAAHelix pomatiaedible snailHPAHomarus americanusCalifornia lobsterHMAIberis amaraIAALaburnum alpinumscotch alburnumLAALens culinarislentilLcHLimax flavusgarden slugLFALimulus polyphemushorseshoe crabLPALotus tetragonolobusasparagus peaLotusLycopersicon esculentumtomatoLEAMaackia amurensismaackiaMAAMaclura pomiferaosage orangeMPAMangifera indicamangoMIANarcissus pseudonarcissusdaffodilNPAPerseau americanaavocadoPAAPhaseolus lunatuslima beanLBAPhaseolus vulgarisred kidney beanPHA-L, PHA-EPHA-P, PHA-MPhaseolus vulgarisblack kidney beanblack beanPhytolacca americanapokeweedPWM, PWAPisum sativumgarden peaPSA, PEAPsophocarpus tetragonolobuswinged beanPTARicinus communiscastor beanRCA-I, RCA-IIRobinia pseudoacaciablack locustRPASalvia horminumsalviaSHASalvia sclareasalviaSSASambucus nigraelderberrySNASolanum tuberosumpotatoSTASophora japonicaJapanese pagoda treeSJATrichosanthes kirilowiitianhuafen, ChinaTKAgourdTrifolium repenswhite cloverRTATriticum vulgarewheat germWGATulipa sptulipTLUlex europaeusgorse or furzeUEA-I, UEA-IIUrtica dioicastinging nettleUDAVicia fabafava bean, broad beanVFAVicia gramineaVGAVicia villosahairy vetchVVAVigna radiatemung beanVRAViscum albummistletoeVAAWisteria floribundaJapanese wisteriaWFA
Possible Lectin Targets.
A number of cell surface carbohydrate moieties are known to undergo both qualitative and quantitative change as cells are pathologically transformed, including in precancers or cancers. Such moieties are potential targets for lectin-based methods of detecting pathological transformation. The following is a non-limiting review of some of the carbohydrate moieties that are known to undergo such changes.
Cell Surface/Blood Group Antigens.
Malignant transformation that results in disordered cell surface carbohydrate expression is often associated with glycosylation changes in the carbohydrates that can include (1) synthesis of new carbohydrate structures and (2) deletion of more complex structures and accumulation of smaller precursor structures. Although originally described as major erythrocyte antigens, blood group antigens are found on epithelial cells of various tissues including oral mucosa.
In mucosal squamous cell carcinoma in the head and neck, especially in oral cancer, incomplete glycosylation of cell surface carbohydrates of the ABO(H) blood group antigens is significant (Dabelsteen et al., Acta Pathol Microbiol Scand. 1988: 17: 506-511; Mandel et al., J Oral Pathol. 1988; 17: 506-511). A-, B-, and H blood group antigens carry their specific antigenic determinants at the ends of their carbohydrate chains. The A or B-antigens and their immediate precursor, H-antigen, are carried by branched or unbranched type 1 ([Galβ1→3GlcNAc]nβ1→3Gal→R) or type 2 ([Galβ1→4GlcNAc]nβ1→3Gal→R) chains, in which Gal is D-galactose and GlcNAc is N-acetyl-D-glucosamine, depending on the type of tissues (e.g. in gastrointestinal epithelia and their secretions, antigens are carried predominantly by type 1 chains, while in erythrocytes they are carried by type 2 chains) (Feske S et al, Nature 2006; 441: 179-85; Endo Y et al, Int J Cancer 2004; 110: 225-31). A and B antigens expressed in normal epithelial cells are lost or diminished, while the precursor H antigen is strongly expressed in the same malignant cells.
In addition, cell surface expression Lewis determinants displayed on the terminus of glycolipids are modified during carcinogenesis and metastasis. The Lewis determinants are structurally related to determinants of the ABO and the H/h blood group systems. They are assembled by sequential addition of specific monosaccharides onto terminal saccharide precursor chains on glycolipids or glycoproteins. The ABH and Lewis glycoproteins possess a common basic structure and their blood group specificity is determined by the sequence and linkage. There are two Lewis antigens, termed Le-a and Le-b. The presence of fucose linked to C-4 of N-acetylglucosamine on a Type 1 chain results in Le-a activity, but a Type 2 oligosaccharide containing fucose linked to C-3 of N-acetylglucosamine on a Type 2 chain results in very weak Le-a activity. The appearance of a second fucose on a type one chain results in the appearance of a new antigenic determinant, Le-b, and the loss of most H and Le-a antigenicity. A Type 2 difucosyl chain has very weak Le-b activity. Since these blood group antigens are associated with carbohydrate side chains of cell surface glycoconjugates, lectins or antibodies could be used to highlight aberrant glycosylation associated with cancerous lesions.
Thomsen-Friedenreich Antigen(s).
The Thomsen-Friedenreich (TF) antigen, a well-defined pan-carcinoma antigen associated with a variety of cancers in different tissues (Monteith G R and Roufogalis B D, Cell Calcium 1995; 18: 459-70), has been targeted recently for the development of tumor selective vaccines (Putney J W Jr, Nat Cell Biol 2009; 11: 381-382). During carcinogenesis, T, Tn and sialyl Tn antigens, core O-linked glycoprotein structures, are formed when core carbohydrate structures are incompletely glycosylated (Saussez S et al., Cancer, 1998; 15:252-260). In oral cancers, the Thomsen-Friedenreich antigen is abundantly expressed in well-differentiated squamous cell carcinomas, and therefore these glycol structures offer possible binding sites for lectins and anti-TF antigen antibodies.
Mucins and Mucin-Like Proteins.
Mucins are large glycoproteins with a “rod-like” conformation caused by the presence of many clustered glycosylated serines and threonines in tandem repeat regions. Most epithelial mucin polypeptides belong to the MUC family. In the normal polarized epithelium, mucins are expressed exclusively on the apical domain, toward the lumen of the organ. Likewise, soluble mucins are secreted exclusively into the lumen. However, the loss of correct topology in malignant epithelial cells allows mucins to be expressed on all aspects of the cells, and soluble mucins can then enter the extracellular space and body fluids such as the blood plasma. In many instances, mucins appear to be the major carriers of altered glycosylation in carcinomas. Another abnormal feature of carcinoma mucins is incomplete glycosylation. Glandular epithelial tissues synthesize and secrete high molecular weight mucin and mucin-like glycoproteins containing O-linked oligosaccharide chains (chains which are branched off of serine or threonine). Under certain pathological conditions, including malignant transformation, the rate of production and degree of glycosylation of these glycoproteins is altered, frequently as a result of incomplete assembly of the normal cell surface structures (Putney J W and Bird G S, J Physiol 2008; 586: 3055-9; Catterall W A et al. Pharmacol Rev 2005; 57: 411-25).
During carcinogenesis, human secretory mucin (MUC) genes can also be misregulated, thereby further affecting mucin levels. MUC1 (polymorphic epithelial mucin, Episialin, MAM-6 antigen, epithelial membrane antigen), a membrane-associated mucin expressed in many secretory epithelia, is expressed at a low level in normal oral mucosa (Gunthorpe M J and Chizh B A, Drug Discov Today 2009; 14: 56-67; Pankratov Y V, Lalo U V and Krishtal O A, J Neurosci 2002; 22: 8363-9). The other MUC genes include MUC2 (prominent in the small and large intestine), MUC3 (predominant in the small intestine), MUC4 (universal for the epithelia), MUC5B (essentially in glandular acini in the submaxillary gland), MUC5C (present in respiratory and gastric tracts) (Putney J W Jr, Nat Cell Biol 2009; 11: 381-3825; Putney J W and Bird G S, J Physiol 2008; 586: 3055-9), MUC6 (prominent in the stomach and gall bladder) (Catterall W A et al, Pharmacol Rev 2005; 57: 411-257) and MUC7 (mainly in the submandibullary gland) (Saponara S, Sgaragli G and Fusi F, Eur J Pharmacol 2008; 598: 75-80). Overexpression of the mucin proteins, especially MUC 1, is associated with many types of cancer.
Given the abundance of simple mucin-type O-glycosylation in oral and other mucosa (Putney J W Jr, Nat Cell Biol 2009; 11: 381-382), one or more novel membrane-associated mucin-like glycoproteins are likely modified by glycosylation and overexpressed during carcinogenesis, and these modifications can be targeted by lectins.
Fucosylation.
Fucosylation (the addition of L-fucose at an oligosaccharide chain terminus) mediates several specific biologic functions. Tumor cells escape recognition by increasing surface fucosylation levels, which leads to decreased adhesion and uncontrolled tumor growth. Monitoring serum/tissue fucose levels could be a promising approach for the early detection, diagnosis, and prognosis of various cancer types. As such, antibodies or lectins selective of L-fucose groups at the end of glycoconjugates could be used as a cancer detection scheme.
Sialylation or Desialylation.
Sialic acid's position at or near non-reducing termini underlies its vital role in determining surface characteristics of cells and secreted glycoproteins. Cell surface glycoconjugate oligosaccharides of cancer patients contain increased levels of the sialic acid, N-acetylneuraminic acid (Neu5Ac or NANA), the predominant sialic acid found in mammalian cells. Accordingly, glycoprotein-bound and glycolipid-bound sialic acid are important cancer biomarkers (Ramsey I S, Delling M and Clapham D E, Annu Rev Physiol 2006; 68: 619-47; Catterall W A et al, Pharmacol Rev 2005; 57: 411-25). Sialic acid content is typically measured either as total sialic acid (TSA; glycoprotein- and glycolipid-bound sialic acid) or as lipid-bound sialic acid (LSA; glycolipid-bound sialic acid) (Saponara S, Sgaragli G and Fusi F, Eur J Pharmacol 2008; 598: 75-80). Serum TSA and LSA levels are significantly elevated in sera of patients with oral and pharyngeal cancer compared with controls (Gunthorpe M J and Chizh B A, Drug Discov Today 2009; 14: 56-673). Additionally, elevated serum sialic acid levels are reported to be correlated with the clinical staging, prognosis and recurrence of malignancies (Pankratov Y V, Lalo U V and Krishtal O A, J Neurosci 2002; 22: 8363-9; Feske S et al, Nature 2006; 441: 179-85).
Although glycoprotein and glycolipid-bound sialic acid content and its correlation to diagnosis and detection of cancer has not been fully studied, lectins selective of sialic acid groups at the end of glycoconjugates may be useful for cancer detection.
Epidermal Growth Factor Receptor (EGFR).
EGFR, a 170 kDa transmembrane glycoprotein (member of the erbB family of cell surface receptors) that plays a role in several metabolic pathways, is composed of 3 major regions: an amino-terminal extracellular ligand-binding domain, a hydrophobic transmembrane domain and a cytoplasmic domain. The extracellular domain, more precisely the ligand-binding domain, is characterized by a relatively high content of carbohydrates that can be glycosylated.
EGFR overexpression is a well-established biomarker in premalignant and invasive oral squamous epithelial lesions. EGFR expression is detected at all stages of carcinogenesis, from normal-early hyperplasia, dysplasia to invasive carcinoma (Monteith G R et al, Nat Rev Cancer 2007; 7: 519-30; Jackson T R et al, Biochem J 1988; 253: 81-618, 19). EGFR expression is elevated during the progression from hyperplasia to dysplasia and increases during progression from dysplasia to invasive squamous cell carcinoma (SCC) (Pacifico F et al. J Mol Endocrinol 2003; 30: 399-40920). Interestingly, Nouri et al. (Int J Cancer 2004; 110: 225-3122) found that 73% of the invasive oral SCC they studied showed strong EGFR expression. Other reports have estimated overexpression of EGFR in all oral cancers at 50-98% (Prasad V et al. Cancer Res 2005; 65: 8655-6123).
Fluorescent dyes conjugated to monoclonal antibodies have been used to label EGFR on the surface of SiHa cervical cancer cells and in ex vivo human oral cavity biopsies (Ramsey I S, Delling M and Clapham D E. Annu Rev Physiol 2006; 68: 619-47). Gold nanoparticles and nanoshells (spherical nanoparticles having a core made of a dielectric material such as zinc selenide, sapphire, or glass, covered by a thin metallic shell) have also been used to target surface receptors in cell lines and ex vivo tissues. For example, nanoshells targeted with anti-Her2 have been used to label SK-BR-3 breast cancer cells in culture (Gunthorpe M J and Chizh B A, Drug Discov Today 2009; 14: 56-67), and gold nanoparticles conjugated to EGFR antibodies have been used to target EGFR on the surface of SiHa cervical cancer cells and in human cervical cancer biopsies (Pankratov Y V, Lalo U V and Krishtal O A. J Neurosci 2002; 22: 8363-9). Most recently, EGFR expression in oral neoplasia was investigated by conjugating an EGF peptide to Alexa-647 (a NIR fluorescent dye) and observing its binding ability on extracted oral cancers (Nitin, et al, Neoplasia 2009; 11:542-551).
In addition to changing EGFR expression, cancer also post-translationally modifies EGFR by phosphorylation, glycosylation, and by forming disulfide bridges. It is likely that all the carbohydrates on the EGFR (approximately 30 kDa) are present as N-linked oligosaccharide chains. Since EGFR is overexpressed in oral cancers, and the extracellular component of EGFR has a predictable post-glycosylation structure, lectins targeting this structure could effectively label EGFR to reveal oral cancer. Furthermore, VEGFR (vascular EGFR) is also overproduced in oral cancer, and could be a target of lectin-based detection. Nevertheless, to the best of our knowledge, EGFR labeling via lectins that specifically target EGFR or VEGFR has not been investigated as a means to detect cancerous tissue in-vitro, ex-vivo, or in-vivo.
In oral cancer, lesions appear as a growth or sore in the mouth that does not go away. Oral cancer, which includes cancers of the lips, tongue, cheeks, floor of the mouth, hard and soft palate, sinuses, and pharynx (throat), is life threatening if not diagnosed and treated early. The most common type of oral cancer is oral neoplasia or oral neoplasm, sometimes referred to as epithelial cancer, which is characterized as squamous cell carcinoma.
Out of the 400,000 individuals newly diagnosed each year with oral cancer, about half will die in the five years after diagnosis (Parekh A B and Putney J W Jr., Physiol Rev 2005; 85: 757-810). This number has not significantly improved over the past few decades, despite continued refinement of surgical techniques and screening technologies. The death rate for oral cancer is particularly high since it is not generally discovered until it has spread to other parts of the body. Because the early signs and symptoms may be misinterpreted by individuals and screening tests tend to be inconsistently employed, oral cancer may not be detected at early stages when it is most treatable. However, early detection and treatment of cancers can markedly decrease mortality rates and increase survival rates.
A patient's primary point of care for oral cancer detection is a general dental practitioner. As part of a routine dental exam, a dentist will conduct an oral cancer screening exam. More specifically, a dentist will feel for any lumps or irregular tissue changes in a patient's neck, head, face, and oral cavity. Recently, several commercially available technologies have been developed (i.e. ViziLite®, VELscope®, Trimira®, etc.) to screen for oral cancer. However, the effectiveness of these technologies to aid in the detection of oral cancers is inconsistent, and it appears these modalities fail to noticeably improve the detection of oral lesions from routinely preformed standard head and neck exams (Putney J W Jr., Nat Cell Biol 2009; 11: 381-382).
For those instances in which a patient has been diagnosed with cancer, the physician generally determines if the cancer can be removed or resected by surgery. Patients who have cancer that has not spread beyond a local area frequently may be treated by completely resecting the tumor. Prior to surgery, various images of the tumor are obtained such as X-rays, CT scans, MRI scans or PET images. Although such images provide guidance for surgery, these images cannot be generated in real time during surgery to guide the surgeon to the tumor.
What is needed are methods, devices and kits for detecting and identifying oral cancer tissue and potential surgical margins that are convenient, cost effective, and that can be used in a variety of clinical settings, including dental offices and operatories.